Cell cycle regulation of the NHEJ DNA double-strand break repair pathway in eukaryotes

Our cells contain DNA, the so called "genetic blueprint of life" which encodes the information for our genes. DNA has a simple repeating structure composed of two complementary strands of DNA, which form a double-helix structure. The integrity of DNA is constantly being challenged by various DNA-damaging agents. These agents include high energy UV and X-ray radiation from the sun, chemicals (both man-made and environmental) and even the oxygen we breathe can attack and damage DNA.

In recent years, it has been realised that our own cells produce a large number of different proteins responsible for repairing this DNA damage which, if left unchecked, would lead to the destabilization of our genomic DNA. It is now recognized that genome instability is one of the major drivers for the development of major human diseases, such as cancer. These protein "machines" can cut out and replace aberrant DNA mutations/structures and splice together broken DNA strands. One such machine is the non homologous end-joining (NHEJ) DNA break repair complex. This repair apparatus is able to detect physical breaks in the DNA helices, bring the ends back together, remodel them if required and, finally, reseal these breaks to restore the continuity of the double-strand DNA. However, if unregulated, an over abundance of these repair machines could potentially also have dangerous consequences by interfering with normal cell processes, such as DNA replication and cell division. The focus of this proposal is to understand how our cells control these repair complexes to ensure they are available when required but, importantly, switched off when they are likely to be harmful to the cell.

DNA double-strand break (DSB) repair is crucial for the maintenance of genomic integrity and loss of this pathway can lead to translocations and carcinogenesis. Two major DSBs pathways are deployed to repair DSBs. In S/G2 phase, homologous recombination (HR) utilizes the homologous chromosome as a template to accurately repair DSBs. In contrast, sister chromatids are not available in G0/G1, where non homologous end-joining (NHEJ) is the predominant break repair pathway. The eukaryotic NHEJ complex consists of a number of core factors: XLF, Ku, XRCC4 and ligase IV (Lig4), which together co-ordinate DSB repair.

Although recent reports have increased our understanding of cell cycle regulation of homologous recombination (HR)-mediated break repair in S/G2 phase, little is known about the regulation of NHEJ pathways in G0/G1. As human cells are predominantly in G1, it is critical that we understand how DSBs repair is regulated in this phase of the cell cycle and the consequences of deregulation, as can occur in cancer cells. In this proposal, we propose to identify the mechanism(s) by which NHEJ is regulated through the cell cycle and address the consequences of deregulation of this crucially important break repair pathway in eukaryotic cells. These studies will also advance our understanding of the basic mechanisms underlying the repair of DSBs by the NHEJ pathway.

This proposal focuses on the fundamental processes of DNA repair in eukaryotic cells. This research will have significant impact on our understanding of repair pathways associated with DNA breaks, as well as contributing to the knowledge of pathways related to its dysfunction e.g. cancer and also exploitation of this pathway to treat related disorders. In addition, characterisation of these basic DNA repair mechanisms will have significant impact on the development of novel biotechnology tools and compounds for drug development that could lead to the discovery of exciting lead compounds for new anti-cancer drugs.

The major areas of impact resulting directly from this research project:

1. Training impacts: This project provides ample opportunities to train researchers to use cutting-edge technologies, outlined in the application, to address fundamental scientific questions that have potentially important clinical and industrial applications These researchers will, in turn, supervise undergraduate and PhD students. This work will therefore significantly impact on the training of future young scientists, who will hopefully go on to set up their own research groups. Their newly acquired skills will also be readily transferable to other scientific areas, including clinical, biotechnology and industrial research, teaching and scientific writing. Training and maintaining a highly skilled scientific work force will significantly impact on the UK's ability to remain a world leader in academic and industrial research.

2. Clinical Impacts: NHEJ has been show to be an important cellular pathway required to maintain DNA stability, in both prokaryotes and eukaryotes, and its loss results in major genomic instability. Cellular deregulation of NHEJ contributes to the development of pre-cancerous cells, suggesting that this pathway could be an excellent drugable target to treat a range of proliferative disorders. The molecular and cellular information obtained from these studies will be of great benefit to those in the clinical/pharma sector to assist in the development of potential lead compounds for future drug development to treat a range of cellular disorders e.g. cancer.

3. Industrial impacts: We have previously patented and are currently developing a molecular biology system, based on this NHEJ DNA repair complex, into an application for the biotechnology sector. We will use this recently acquired experience to optimally exploit the yeast NHEJ proteins, which also have excellent potential for commercial development for uses in the biotechnology sector e.g. uses in cloning and related molecular biology applications. Notably, this system may also aid our development of small molecular inhibitors of NHEJ pathways that could potentially act as lead compounds for a future drug development programme.